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Cat. No.: FUAK-100 (for 100T)

Description

Fumarate Assay Kit is based on a WST-8 colorimetric reaction that allows for the rapid and highly sensitive detection of fumarate and fumarate salts in biological fluids such as tissue or cell samples, serum, and plasma using a colorimetric method.

Fumaric acid, also known as fumaric acid, trans-butenedioic acid, fumeric acid, or trans-2-butenedioic acid, is the trans isomer of maleic acid with a molecular formula of C4H4O5 and a molecular weight of 116.07. Fumaric acid is the simplest unsaturated dicarboxylic acid and was first discovered in fumarole. It is also found in high concentrations in various mushrooms, plant tissues, and fresh beef. Fumaric acid is one of the most widely used feed additives and acidulants, and it also serves as an effective anti-stress agent and preservative. It is one of the most potent solid acids, with an acidity 2.5 times that of citric acid, and it has better emulsifying and stabilizing properties. It is often used as a substitute for malic acid, citric acid, or benzoic acid in food additives. Fumaric acid is widely used as an acidity regulator, acidulant, mold inhibitor, preservative, antioxidant, pickling agent, and flavoring agent in the processing of meat products, fish products, and other food and beverage items.

As an important intermediate in the tricarboxylic acid cycle (TCA cycle), fumaric acid plays a crucial role in metabolism. It participates in a series of biochemical reactions including metabolic processes, tissue structure formation, and enzyme activity in animals. In mammalian liver, fumarate salts are also products of the urea cycle, and upon release into the cytoplasm, they are converted into malate salts, which are then further converted into oxaloacetate while producing NADH in the cytoplasm.

Components

• Buffer A for Metabolic Assay
• Fumarate Assay Buffer
• Enzyme Solution A
• Enzyme Solution B
• Color Development Solution
• Substrate
• Fumarate Standard Solution (10mM)

Principle

Fumaric acid is converted into L-malate under the action of fumarase. The generated L-malate is then oxidized to oxaloacetic acid (OAA) by malate dehydrogenase (MDH), with NAD+ being reduced to NADH during this reaction. The produced NADH then reduces WST-8 to form an orange-yellow formazan, with a maximum absorption peak around 450 nm, in the presence of the electron-coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate). The amount of formazan generated in the reaction system is proportional to the content of fumaric acid in the sample.

Features

• This assay kit offers high sensitivity, a wide linear range, and requires a small sample volume. It can detect fumaric acid concentrations as low as 20 μM with a sample volume of 20 µl, and shows a good linear relationship within the 20-500 μM (0.4-10 nmol) concentration range. The kit includes a fumaric acid standard solution, allowing for the quantification of fumaric acid content in samples by establishing a standard curve.

• This assay kit offers a certain degree of versatility. The cell or tissue samples lysed with the provided lysis buffer can also be used for detection in other metabolic assay kits from the same manufacturer that use the same buffer, making it highly versatile. Additionally, it can be used for protein concentration detection, SDS-PAGE, or Western blotting of proteins that are easily soluble.

• The kit is flexible in use, quick in detection, and has a broad application range. It is suitable for testing biological fluids such as serum, plasma, and urine from mice, rats, humans, as well as cell culture supernatants and tissue or cell samples. The entire process can be completed in approximately 0.5-1 hour. The kit is suitable not only for small sample volumes but also for high-throughput screening in automated systems.

Storage

Store at -20ºC, valid for one year. Color Development Solution and Substrate must be protected from light.

Precautions

• Buffer A for Metabolic Assay, Fumarate Assay Buffer (hereafter referred to as detection buffer), Color Development Solution, and Substrate need to be completely thawed and equilibrated to room temperature before use, otherwise, the detection results may be affected. Enzyme Solutions and Fumarate Standard Solution should be handled on ice.
• Substrate may precipitate during thawing from -20ºC, but the precipitated part will dissolve again once equilibrated to room temperature and will not affect the detection results.
• If serum or other samples are stored at 4ºC, the storage time should not exceed 2 weeks, as this may affect the accuracy of the detection results. Typically, serum samples should be stored at -20ºC, with -80ºC being even better.
• To reduce errors caused by background from the diluent, the diluent for samples and standards should be chosen based on the type of sample. For samples lysed with Buffer A for Metabolic Assay, use Buffer A for Metabolic Assay for dilution. For other samples such as blood, use the detection buffer for dilution.
• This product is for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, nor for use in food or pharmaceuticals, and should not be stored in a residential setting.
• For your safety and health, please wear lab coats and disposable gloves during handling.